Tm2 blocks the ubiquitination of RhoA by Smurf1. (A) GFP–Tm1 and GFP–Tm2 interact with dominant-active (G14V) and more strongly with dominant-negative (T19N) RhoA but not with the negative control GFP–sui (GFP control). GFP–Tm1 and GFP–Tm2 do not interact with FLAG–Cdc42 (FLAG control) also serving as negative control. (B) Immobilized GST–RhoA(T19N) but not GST alone directly binds to purified FLAG–Smurf1. In the presence of increasing amounts of FLAG–Tm2, the binding of RhoA to Smurf1 is gradually lost, and concomitantly increased binding of Tm2 to RhoA is detected. In the presence of 1 μg of FLAG–Tm2, only Tm2 binds to RhoA. (C) FLAG–RhoA is ubiquitinated by wild-type (WT) but not by catalytically inactive (CA) Myc–Smurf1 in transfected HEK293 cells expressing GFP as revealed by anti-HA immunoblotting. GFP–Tm2 and GFP–Synaptopodin (Synpo) but not GFP–Tm1 or GFP–α-actinin-4 (Act-4) markedly reduces the ubiquitination of RhoA. No RhoA ubiquitination is detected in the absence of HA–ubiquitin. (D) Synaptopodin and Tm differ in their protein half-life. Treatment of podocytes with cycloheximide results in the rapid degradation of endogenous synaptopodin with a calculated half-life of 17.1±2.4 h. In contrast, Tm protein abundance remains stable over the entire 24-h observation period. (E) An extended model for the regulation of RhoA signalling. In the canonical model of the Rho GTPase cycle, the activity of Rho GTPases is regulated by a switch between an inactive GDP-bound and an active GTP-bound form. The cycle is tightly regulated mainly by guanine exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine dissociation inhibitors (GDIs). Tm and synaptopodin promote stress fibre formation through the inhibition of Smurf1-mediated ubiquitination of RhoA. Thus, repression of Smurf1-induced proteasomal degradation of RhoA by structurally unrelated actin-binding proteins is a conserved mechanism for the regulation of RhoA signalling, in addition to the GDP/GTP switch.